2. The article of claim 1, wherein the article is produced by a process
of:diluting one or more monomers in a volatile organic solvent;depositing
the diluted monomers on the substrate surface of the article;evaporating
substantially all of the solvent; andpolymerizing the monomers to form
the polymer layer attached to the surface.

3. The article of claim 2 wherein the volatile solvent is ethanol.

4. The article of claim 1 wherein the polymer material disposed on the
surface of the article has a surface area greater than about 5 mm.sup.2.

5. The article of claim 1, further comprising a well, wherein the surface
of the substrate on which the polymer material is disposed is in the
well.

6. The article of claim 5, wherein the article is selected from the group
consisting of a multi-well plate, a petri dish, a beaker, a tube, and a
flask.

Description:

PRIORITY

[0001]This application claims priority to U.S. provisional Appl. No.
61/063,010, filed Jan. 30, 2008, and is incorporated by reference in its
entirety.

FIELD

[0002]The present disclosure relates to cell culture articles and methods
of use thereof, and more particularly to articles for supporting the
culture of stem cell derived oligodendrocyte progenitor cells.

BACKGROUND

[0003]Pluripotent stem cells, such as human embryonic stem cells (hESCs)
have the ability to differentiate into any of the three germ layers,
giving rise to any adult cell type in the human body. This unique
property provides a potential for developing new treatments for a number
of serious cell degenerative diseases, such as diabetes, spinal cord
injury, heart disease and the like. For example, spinal cord damage is
generally irreversible with current treatments, leaving approximately
250,000 Americans in a devastating position. However, as stem cell
research has developed, new exciting possibilities have arisen for people
suffering from spinal cord injury. ES cell-derived neural cells have been
used by researchers to treat nervous system disorders in animal models.
In earlier work, researchers showed that mouse ES cells could be
stimulated to differentiate into neural cells that, when transplanted
into mice with neurological disorders, helped to restore normal function.

[0004]However there remain obstacles in the development of such hESC-based
treatments. Such obstacles include obtaining and maintaining adequate
numbers of undifferentiated hESCs in tissue culture and controlling their
differentiation in order to produce specific cell types. Stem cell
cultures, such as hESC cell cultures are typically seeded with a small
number of cells from a cell bank or stock and then amplified in the
undifferentiated state until differentiation is desired for a given
therapeutic application To accomplish this, the hESC or their
differentiated cells are currently cultured in the presence of surfaces
or media containing animal-derived components, such as feeder layers,
fetal bovine serum, or MATRIGEL®. These animal-derived additions to
the culture environment expose the cells to potentially harmful viruses
or other infectious agents which could be transferred to patients or
compromise general culture and maintenance of the hESCs. In addition,
such biological products are vulnerable to batch variation, immune
response and limited shelf-life.

[0005]Some steps have been taken to culture hESCs either in media or on
surfaces that are free of animal-derived components. However, the
response of hESCs or their differentiated derivatives is difficult to
predict as components of the surface or culture medium change. Yet some
advances have been made. For example, hESC-derived oligodendrocyte
progenitor cells (OPCs) have been cultured in defined serum-free medium.
While such culture systems are not completely xeno-free culture systems
when the matrices employed contain animal-derived components, such as
gelatin and MATRIGEL, they do provide a step toward the eventual clinical
application of hESC-derived OPCs. By way of further example, some
synthetic surfaces have been identified that can support differentiation
of human epithelial stem cells into epithelial cells. However, the
systems employed relied on serum medium for the cell culture, which still
potentially causes problem as described before for all biological animal
derived components. To date, a completely animal free system employing a
chemically defined medium and a synthetic surface has not yet been
identified for culturing stem cells or cells derived from stem cells.

[0007]In an embodiment, a method for culturing oligodendrocyte progenitor
cells is provided. The method includes depositing a suspension containing
the oligodendrocyte progenitor cells on a polymer material and culturing
the deposited oligodendrocyte progenitor cells in a cell culture medium.
The polymer material comprises a homopolymer or copolymer of selected one
or more acrylate monomers.

[0008]In an embodiment, a culture of oligodendrocyte progenitor cells is
provided. The culture includes an article having a polymer material
disposed on a surface. The culture further includes the oligodendrocyte
progenitor cells disposed on the polymer material and a culture medium in
which the oligodendrocyte progenitor cells are cultured. The polymer
material comprises a homopolymer or copolymer of selected one or more
acrylate monomers.

[0009]In an embodiment, a cell culture article for culturing
oligodendrocyte progenitor cells in a chemically defined medium is
provided. The article includes a substrate having a surface and a polymer
material disposed on the surface. The polymer material comprises a
homopolymer or copolymer of selected one or more acrylate monomers.

[0010]One or more of the various embodiments presented herein provide one
or more advantages over prior surfaces for culturing stem cell-derived
OPCs. For example, the synthetic surfaces reduce potential contamination
issues associated with surfaces having components obtained from or
derived from animal sources. Such surfaces may also provide for improved
shelf life compared to those surfaces with biological components. The
ability to culture stem cell-derived OPCs in chemically-defined media
further reduces potential contamination issues. In addition, there will
likely be less batch to batch variation in the ability of the synthetic
surfaces or chemically defined media, resulting in improved
reproducibility of culture results and expectations. These and other
advantages will be readily understood from the following detailed
descriptions when read in conjunction with the accompanying drawings.

[0014]FIGS. 4A-C are fluorescence images of immunostained hES cell-derived
OPCs after replating on acrylate coated surface 123-2 (A), 122-3 (B) and
Matrigel® (C) between 28-day and 35-day as well as between 35-day and
42-day. OPCs derived from human ES cells were immunostained for the OPC
marker, Olig 1 (green) and nestin (red).

[0016]The drawings are not necessarily to scale. Like numbers used in the
figures refer to like components, steps and the like. However, it will be
understood that the use of a number to refer to a component in a given
figure is not intended to limit the component in another figure labeled
with the same number. In addition, the use of different numbers to refer
to components is not intended to indicate that the different numbered
components cannot be the same or similar.

DETAILED DESCRIPTION

[0017]In the following detailed description, reference is made to the
accompanying drawings that form a part hereof, and in which are shown by
way of illustration several specific embodiments of devices, systems and
methods. It is to be understood that other embodiments are contemplated
and may be made without departing from the scope or spirit of the present
disclosure. The following detailed description, therefore, is not to be
taken in a limiting sense.

[0018]All scientific and technical terms used herein have meanings
commonly used in the art unless otherwise specified. The definitions
provided herein are to facilitate understanding of certain terms used
frequently herein and are not meant to limit the scope of the present
disclosure.

[0019]As used in this specification and the appended claims, the singular
forms "a", "an", and "the" encompass embodiments having plural referents,
unless the content clearly dictates otherwise. As used in this
specification and the appended claims, the term "or" is generally
employed in its sense including "and/or" unless the content clearly
dictates otherwise.

[0020]Unless stated otherwise, ratios of compounds in a composition, such
as a solution, are stated on a by volume basis.

[0021]As used herein, "have", "having", "include", "including",
"comprise", "comprising" or the like are used in their open ended sense,
and generally mean "including, but not limited to".

[0022]The present disclosure describes, inter alia, articles having
synthetic surfaces for culturing stem cell-derived OPCs and methods for
culturing stem cell-derived OPCs on such surfaces. In some embodiments,
the synthetic surfaces are used in combination with a chemically defined
medium to culture stem cell-derived OPCs. The surfaces may be useful in
differentiating stem cells, such as hESCs, into OPCs or for proliferating
such stem cell-derived OPCs.

1. Cell Culture Article

[0023]Referring to FIG. 1, a schematic diagram of article 100 for
culturing cells is shown. The article 100 includes a base material
substrate 10 having a surface 15. A synthetic polymer coating layer 20 is
disposed on the surface 15 of the base material 10. While not shown, it
will be understood that synthetic polymer coating 20 may be disposed on a
portion of base material 10. The base material 10 may be any material
suitable for culturing cells, including a ceramic substance, a glass, a
plastic, a polymer or co-polymer, any combinations thereof, or a coating
of one material on another. Such base materials 10 include glass
materials such as soda-lime glass, pyrex glass, vycor glass, quartz
glass; silicon; plastics or polymers, including dendritic polymers, such
as poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate),
poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane)
monomethacrylate, cyclic olefin polymers, fluorocarbon polymers,
polystyrenes, polypropylene, polyethyleneimine; copolymers such as
poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic
anhydride), poly(ethylene-co-acrylic acid) or derivatives of these or the
like.

[0025]Synthetic polymer coating 20 provides a surface 25 on which cells
may be cultured. The synthetic polymer surface 20 includes polymerized
acrylate monomers, selected from the group of monomers provided in Table
1 below. Other materials (not shown), such as peptides, may be
incorporated into or conjugated to synthetic polymer surface to produce a
biomimetic surface.

[0026]The acrylates listed in Table 1 may be synthesized as known in the
art or obtained from a commercial vendor, such as Polysciences, Inc.,
Sigma Aldrich, Inc., and Sartomer, Inc.

[0027]As shown in FIG. 1B, an intermediate layer 30 may be disposed
between surface 15 of base material 10 and the synthetic polymer coating
20. Intermediate layer 30 may be configured to improve binding of coating
20 to substrate 10, to facilitate monomer spreading, to render portions
of the surface 10 that are uncoated cytophobic to encourage cell growth
on coated areas, to provide a substrate compatible with a monomer or
solvent where the monomer or solvent is incompatible with the base
material 10, to provide topographical features if desired through, for
example, patterned printing, or the like. For example, if substrate 10 is
a glass substrate, it may be desirable to treat a surface of the glass
substrate with a silane molecule or an epoxy coating. For various polymer
base materials 10 it may be desirable to provide an intermediate layer 30
of polyamide, polyimide, polypropylene, polyethylene, or polyacrylate.
While not shown, it will be understood that synthetic polymer coating 20
may be disposed on a portion of intermediate layer 30. It will be further
understood that intermediate layer 30 may be disposed on a portion of
base material 10.

[0028]In various embodiments, surface 15 of base material 10 is treated,
either physically or chemically, to impart a desirable property or
characteristic to the surface 15. For example, and as discussed below,
surface 15 may be corona treated or plasma treated. Examples of vacuum or
atmospheric pressure plasma include radio frequency (RF) and microwave
plasmas both primary and secondary, dielectric barrier discharge, and
corona discharge generated in molecular or mixed gases including air,
oxygen, nitrogen, argon, carbon dioxide, nitrous oxide, or water vapor.

[0029]Synthetic polymer coating layer 20, whether disposed on an
intermediate layer 30 or base material 10, preferably uniformly coats the
underlying substrate. By "uniformly coated", it is meant that the layer
20 in a given area, for example a surface of a well of a culture plate,
completely coats the area at a thickness of about 5 nm or greater. While
the thickness of a uniformly coated surface may vary across the surface,
there are no areas of the uniformly coated surfaces through which the
underlying layer (either intermediate layer 30 or base material 10) is
exposed. Cell responses across non-uniform surfaces tend to be more
variable than cell responses across uniform surfaces.

[0030]Synthetic polymer coating layer 20 may have any desirable thickness.
However, it has been found that thicker coatings, e.g. coatings of
greater than about 10 micrometers, tend to have unevenness around the
periphery of the coating due to surface tension. In various embodiments,
the thickness of the coating layer 20 is less than about 10 micrometers.
For example, the thickness may be less than about 5 micrometers, less
than about 2 micrometers, less than about 1 micrometers, less than about
0.5 micrometers or less than about 0.1 micrometers.

[0031]The polymer material forming synthetic polymer layer 20 may be
cross-linked to any suitable degree. Higher degrees of cross-linking may
result in reduced waste product and reduced cell toxicity.

[0032]Article 100, in numerous embodiments, is cell culture ware having a
well, such as a petri dish, a multi-well plate, a flask, a beaker or
other container having a well. Referring now to FIG. 2, article 100
formed from base material 10 may include one or more wells 50. Well 50
includes a sidewall 55 and a surface 15. Referring to FIG. 2B-C, a
synthetic polymer coating 20 may be disposed on surface 15 or sidewalls
55 (or, as discussed above with regard to FIG. 1 one or more intermediate
layer 30 may be disposed between surface 15 or sidewall 55 and synthetic
polymer coating 20) or a portion thereof.

[0033]In various embodiments, article 100 includes a uniformly coated
layer 20 having a surface 25 with an area greater than about 5 mm2.
When the area of the surface 15 is too small, reliable cell responses may
not be readily observable because some cells, such as human embryonic
stem cells, are seeded as colonies or clusters of cells (e.g., having a
diameter of about 0.5 mm) and adequate surface is desirable to ensure
attachment of sufficient numbers of colonies to produce a quantitative
cell response. In numerous embodiments, an article 100 has a well 50
having a uniformly coated surface 15, where the surface 15 has an area
greater than about 0.1 cm2, greater than about 0.3 cm2, greater
than about 0.9 cm2, or greater than about 1 cm2.

2. Coating of Synthetic Polymer Layer

[0034]A synthetic polymer layer may be disposed on a surface of a cell
culture article via any known or future developed process. Preferably,
the synthetic polymer layer provides a uniform layer that does not
delaminate during typical cell culture conditions. The synthetic polymer
surface may be associated with the base material substrate via covalent
or non-covalent interactions. Examples of non-covalent interactions that
may associate the synthetic polymer surface with the substrate include
chemical adsorption, hydrogen bonding, surface interpenetration, ionic
bonding, van der Waals forces, hydrophobic interactions, dipole-dipole
interactions, mechanical interlocking, and combinations thereof.

[0035]In various embodiments, the base material substrate surface is
coated according to the teachings of co-pending application Ser. No.
______, filed on even date herewith, naming Gehman et al. as inventors,
having attorney docket number 20726, and entitled STEM CELL CULTURE
ARTICLE AND SCREENING, which is hereby incorporated herein by reference
in its entirety for all purposes to the extent that it does not conflict
with the disclosure presented herein.

[0036]In numerous embodiments, monomers are deposited on a surface of a
cell culture article and polymerized in situ. In such embodiments, the
base material will be referred to herein as the "substrate" on which the
synthetic polymer material is deposited. Polymerization may be done in
solution phase or in bulk phase.

[0037]As many of the monomers identified in Table 1 above are viscous, it
may be desirable to dilute the monomers in a suitable solvent to reduce
viscosity prior to being dispensed on the surface. Reducing viscosity may
allow for thinner and more uniform layers of the synthetic polymer
material to be formed. One of skill in the art will be able to readily
select a suitable solvent. Preferably the solvent is compatible with the
material forming the cell culture article and the monomers. It may be
desirable to select a solvent that is non-toxic to the cells to be
cultured and that does not interfere with the polymerization reaction.
Alternatively, or in addition, selection of a solvent that can be
substantially completely removed or removed to an extent that it is
non-toxic or no longer interferes with polymerization may be desirable.
In additional embodiments, it may be desirable to select solvents which
do not interact with the substrate. Further, it may be desirable that the
solvent be readily removable without harsh conditions, such as vacuum or
extreme heat. Volatile solvents are examples of such readily removable
solvents. As described in co-pending application Ser. No. ______, ethanol
may be a particularly suitable solvent when it is desired to remove
solvent prior to polymerization.

[0038]The monomers may be diluted with solvent by any suitable amount to
achieve the desired viscosity and monomer concentration. Generally the
monomer compositions used according to the teachings presented herein
contain between about 0.1% to about 99% monomer. By way of example, the
monomer may be diluted with an ethanol solvent to provide a composition
having between about 0.1% and about 50% monomer, or from about 0.1% to
about 10% monomer by volume. The monomers may be diluted with solvent so
that the polymer layer 20 achieves a desired thickness. As discussed
above, if the deposited monomers are too thick, an uneven surface may
result. As described in further details in the Examples, uneven surfaces
may be observed when the monomer-solvent composition is deposited on a
surface 15 of a well 50 at a volume of greater than about 8 microliters
per square centimeter of the surface 15. In various embodiments, the
monomer-solvent compositions are deposited on a surface 15 of a well 50
in a volume of about 7 microliters or less per square centimeter of the
surface 15. For example, the monomer-solvent compositions may be
deposited on a surface 15 of a well 50 in a volume of about 5 microliters
or less per square centimeter of the surface 15, or about 2 microliters
or less per square centimeter of the surface 15.

[0039]In various embodiments, article 100 includes a uniformly coated
layer 20 having a surface 25 with an area greater than about 5 mm2.
When the area of the surface 15 is too small, reliable cell responses may
not be readily observable because some cells, such as human embryonic
stem cells, are seeded as colonies or clusters of cells (e.g., having a
diameter of about 0.5 mm) and adequate surface is desirable to ensure
attachment of sufficient numbers of colonies to produce a quantitative
cell response. In numerous embodiments, an article 100 has a well 50
having a uniformly coated surface 15, where the surface 15 has an area
greater than about 0.1 cm2, greater than about 0.3 cm2, greater
than about 0.9 cm2, or greater than about 1 cm2.

[0040]In various embodiments, synthetic polymer surface is deposited on a
surface of an intermediate layer that is associated with the base
material via covalent or non-covalent interactions, either directly or
via one or more additional intermediate layers (not shown). In such
embodiments, the intermediate layer will be referred to herein as the
"substrate" onto which the synthetic polymer surface is deposited.

[0041]In various embodiments, the surface of the base material is treated.
The surface may be treated to improve binding of the synthetic polymer
surface to the base material surface, to facilitate monomer spreading on
the base material surface, or the like. Of course, the base material may
be treated for similar purposes with regard to an intermediate layer. In
various embodiments, the surface is corona treated or vacuum plasma
treated. High surfaces energy obtainable from such treatments may
facilitate monomer spreading and uniform coating. Examples of vacuum
plasma treatment that may be employed include microwave vacuum plasma
treatments and radio frequency vacuum plasma treatments. The vacuum
plasma treatments may be performed in the presence of reactive gases,
such as oxygen, nitrogen, ammonia or nitrous oxide.

[0042]To form the synthetic polymer surface, one or more monomers
presented in Table 1 above are polymerized. If one monomer is used, the
polymer will be referred to as a homopolymer of the monomer. If two or
more different monomers are used, the polymer will be referred to as a
copolymer of the monomers. The monomers employed may be monofunctional,
difunctional, or higher-functional. When two or more monomers are used,
the ratio of the monomers may be varied. In various embodiments, two
monomers are used and the ratio, by volume of the first monomer to the
second monomer ranges from between about 5:95 to about 95:5. For example,
the ratio of the first monomer to the second monomer ranges from between
about 10:90 to about 90:10, about 20:80 to about 80:20, from about 30:70
to about 70:30. In some embodiments, the ratio of the first monomer to
the second monomer is about 50:50, 30:70, or 10:90. It will be understood
that the degree of cross-linking of the polymer may be controlled by
varying the concentration of monomers or the ratios of difunctional or
higher-functional monomers to monofunctional monomers. Increased
concentrations of difunctional or higher-functional monomers will
increase the degree of cross-linking in the chains.

[0043]In addition to the monomers that form the polymer layer, a
composition forming the layer may include one or more additional
compounds such as surfactants, wetting agents, photoinitiators, thermal
initiators, catalysts, activators, and cross-linking agents.

[0044]Any suitable polymerization initiator may be employed. One of skill
in the art will readily be able to select a suitable initiator, e.g. a
radical initiator or a cationic initiator, suitable for use with the
monomers listed in Table 1. In various embodiments, UV light is used to
generate free radical monomers to initiate chain polymerization.

[0046]A photosensitizer may also be included in a suitable initiator
system. Representative photosensitizers have carbonyl groups or tertiary
amino groups or mixtures thereof. Photosensitizers having a carbonyl
group include benzophenone, acetophenone, benzil, benzaldehyde,
o-chlorobenzaldehyde, xanthone, thioxanthone, 9,10-anthraquinone, and
other aromatic ketones. Photosensitizers having tertiary amines include
methyldiethanolamine, ethyldiethanolamine, triethanolamine,
phenylmethyl-ethanolamine, and dimethylaminoethylbenzoate. Commercially
available photosensitizers include QUANTICURE ITX, QUANTICURE QTX,
QUANTICURE PTX, QUANTICURE EPD from Biddle Sawyer Corp.

[0047]In general, the amount of photosensitizer or photoinitiator system
may vary from about 0.01 to 10% by weight.

[0048]Examples of cationic initiators include salts of onium cations, such
as arylsulfonium salts, as well as organometallic salts such as ion arene
systems.

[0049]In various embodiments where the monomers are diluted in solvent
before being deposited on the substrate surface, the solvent is removed
prior to polymerizing. The solvent may be removed by any suitable
mechanism or process. As described in copending application Ser. No.
______, it has been found that removal of substantially all of the
solvent prior to curing, allows for better control of curing kinetics and
the amount of monomer converted. When conversion rates of the monomers
are increased, waste generation and cytotoxicity are reduced.

[0050]Whether polymerized in bulk phase (substantially solvent free) or
solvent phase, the monomers are polymerized via an appropriate initiation
mechanism. Many of such mechanisms are well known in the art. For
example, temperature may be increased to activate a thermal initiator,
photoinitiators may be activated by exposure to appropriate wavelength of
light, or the like. According to numerous embodiments, the monomer or
monomer mixture is cured using UV light. The curing preferably occurs
under inert gas protection, such as nitrogen protection, to prevent
oxygen inhibition. Suitable UV light combined with gas protection may
increase polymer conversion, insure coating integrity and reduce
cytotoxicity.

[0051]The cured synthetic polymer layer may be washed with solvent one or
more times to remove impurities such as unreacted monomers or low
molecular weight polymer species. In various embodiments, the layer is
washed with an ethanol solvent, e.g. 70% ethanol, greater than about 90%
ethanol, greater than about 95% ethanol, or greater than about 99%
ethanol. Washing with an ethanol solvent may not only serve to remove
impurities, which may be cytotoxic, but also can serve to sterilize the
surface prior to incubation with cells.

3. Incubating Cells on Synthetic Polymer Layer

[0052]Stem cell-derived OPCs may be cultured on a synthetic polymer layer,
as described above, according to any suitable protocol. As used herein,
"stem cell derived OPC" means an OPC obtained from differentiation of a
stem cell. In some embodiments, the stem cells are multipotent,
totipotent, or pluripotent stem cells. The stem cells may be present in
an organ or tissue of a subject. In numerous embodiments, the stem cells
are embryonic stem cells, such as human embryonic stem cells. As used
herein, "OPC" or "oligodendrocyte progenitor cell" means precursor cells
to myelin-forming oligodendrocytes.

[0053]Because human embryonic stem cells (hESC) have the ability to grown
continually in culture in an undifferentiated state, the hESC for use in
this invention may be obtained from an established cell line. Examples of
human embryonic stem cell lines that have been established include, but
are not limited to, H1, H7, H9, H13 or H14 (available from WiCell
established by the University of Wisconsin) (Thompson (1998) Science
282:1145); hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen, Inc., Athens, Ga.);
HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (from ES Cell International,
Inc., Singapore); HSF-1, HSF-6 (from University of California at San
Francisco); I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3, J 3.2 (derived at
the Technion-Israel Institute of Technology, Haifa, Israel); UCSF-1 and
UCSF-2 (Genbacev et al., Fertil. Steril. 83(5):1517-29, 2005); lines HUES
1-17 (Cowan et al., NEJM 350(13):1353-56, 2004); and line ACT-14
(Klimanskaya et al., Lancet, 365(9471):1636-41, 2005). Embryonic stem
cells used in the invention may also be obtained directly from primary
embryonic tissue. Typically this is done using frozen in vitro fertilized
eggs at the blastocyst stage, which would otherwise be discarded.

[0054]OPCs according to the invention may also be differentiated from
induced primate pluripotent stem (iPS) cells. iPS cells refer to cells,
obtained from a juvenile or adult mammal, such as a human, that are
genetically modified, e.g., by transfection with one or more appropriate
vectors, such that they are reprogrammed to attain the phenotype of a
pluripotent stem cell such as an hESC. Phenotypic traits attained by
these reprogrammed cells include morphology resembling stem cells
isolated from a blastocyst as well as surface antigen expression, gene
expression and telomerase activity resembling blastocyst derived
embryonic stem cells. The iPS cells typically have the ability to
differentiate into at least one cell type from each of the primary germ
layers: ectoderm, endoderm and mesoderm and thus are suitable for
differentiation into OPCs. The iPS cells, like hESC, also form teratomas
when injected into immuno-deficient mice, e.g., SCID mice. (Takahashi et
al., (2007) Cell 131(5):861; Yu et al., (2007) Science 318:5858).

[0055]Stem cell derived OPCs may be obtained by any suitable methods. One
way to obtain such cells is described in Zhang et al., "Oligodendrocyte
progenitor cells derived from human embryonic stem cells express
neurotrophic factors, Stem Cells and Development, 15: 943-952 (2006),
citing Nistor et al., Human embryonic stem cells differentiate into
oligodendrocytes in high purity and mylenate after spinal cord
transplantation, Glia 49: 385-396 (2005). Briefly, undifferentiated human
embryonic stem cells, such as those derived from the H1 or H7 human
embryonic stem cell lines, may be cultured on MATRIGEL-coated plates in
mouse embryonic fibroblast (MEF) conditioned medium (CM) supplemented
with about 8 ng/ml fibroblast growth factor-2 (FGF-2) or in a chemically
defined medium, such as X-VIVO 10 from Cambrex, supplemented with about
80 ng/ml FGF-2 and 0.5 ng/ml transforming growth factor-β1
(TGF-β1). To induce differentiation, the protocol described by
Nistor et al. may be employed. Briefly, the human embryonic stem cells
may be collagenase digested, scraped, and cultured in defined medium
supplemented with insulin, transferrin, progesterone, putrescin,
selenium, triiodothyroidin and B27 for 28 days on an ultra-low-attachment
plate. The cells may then be cultured in the defined medium for an
additional 14 days on growth-factor reduced MATRIGEL. The cells may then
be treated with FGF-2, epidermal growth factor (EGF) and all-trans
retinoic acid on specified days during differentiation. Differentiation
may occur over a number of days, such as 42 days. Of course, any other
suitable method may be employed.

[0056]Prior to seeding cells, the cells may be harvested and suspended in
a suitable medium, such as a growth medium in which the cells are to be
cultured once seeded onto the surface. For example, the cells may be
suspended in and cultured in serum-containing medium, a conditioned
medium, or a chemically-defined medium. As used herein,
"chemically-defined medium" means cell culture media that contains no
components of unknown composition. Chemically defined media may, in
various embodiments, contain no proteins, hydrosylates, or peptides of
unknown composition. In some embodiments, conditioned media contains
polypeptides or proteins of known composition, such as recombinant growth
hormones. Because all components of chemically-defined media have a known
chemical structure, variability in culture conditions and thus cell
response can be reduced, increasing reproducibility. In addition, the
possibility of contamination is reduced. Further, the ability to scale up
is made easier due, at least in part, to the factors discussed above.

[0057]The cells may be seeded at any suitable concentration. Typically,
the cells are seeded at about 10,000 cells/cm2 of substrate to about
500,000 cells/cm2. For example, cells may be seeded at about 50,000
cells/cm2 of substrate to about 150,000 cells/cm2. However,
higher and lower concentrations may readily be used. The incubation time
and conditions, such as temperature CO2 and O2 levels, growth
medium, and the like, will depend on the nature of the cells being
cultured and can be readily modified. The amount of time that the cells
are incubated on the surface may vary depending on the cell response
being studied or the cell response desired.

[0058]Any suitable method may be used, if desired, to confirm that the
stem cell derived OPCs are indeed OPCs or that the stem cells employed
have successfully differentiated into OPCs. For example, the presence of
certain OPC-selective markers may be investigated. Such markers include
Nestin, Oligo1, platelet derived growth factor receptor alpha
(PDGFRα) and NG2. Antibodies to such markers may be used in
standard immunocytochemical or flow cytometry techniques. In addition or
alternatively, cellular morphology or production of growth factors
detectable in the medium may be evaluated. For example, cultured OPCs may
produce one or more of activin A, HGF, midkine, and TGF-β2, which
may be detectable in the culture medium via standard assays, such as
ELISA.

[0059]The cultured stem cell derived OPCs may be used for any suitable
purpose, including investigational studies in culture, in animals, for
developing therapeutic uses, or for therapeutic purposes. One potential
therapeutic or investigational purpose is repairing damage due to spinal
cord injury.

[0060]In the following, non-limiting examples are presented, which
describe various embodiments of the articles and methods discussed above.

[0061]Acrylic coating surfaces were prepared from homomonomers or
copolymers of various acrylate monomers. For copolymers two different
acrylate monomers were used. A total of 24 homopolymer and 552 copolymer
combinations were applied in wells. Briefly, the monomers were diluted in
ethanol, and IRGACURE 819 photoinitiator to the ratio of 1:9:0.01
(monomer[volume]/ethanol[volume]/photoinitiator[weight]) to prepare the
formulation. For copolymers, two different monomers were mixed with the
volume ratio of 70:30 or 30:70. In copolymer formulation, total
monomers[volume]/ethanol[volume]/photoinitiator[weight] still remain the
ratio of 1:9:0.01. The formulations were placed in a well of a plasma
treated cyclic olefin copolymer 96 well plates (provided by Corning Life
Science development group) at a volume of 5 μL using BioTek Precession
Microplate Pipetting System. Each well received a predetermined
homopolymer or copolymer combination, with some wells being coated with
MATRIGEL as a positive control. For the wells coated with acrylate
monomers, the ethanol solvent was removed by evaporation at room
temperature for 3 hr, which removes >99% of the ethanol. The coatings
were then cured with 13 mW/cm2 pulsed (100 Hz) UV light (Xenon
RC-801) for 1 min in N2 purged box (with fused silica window). After
curing, a washing step was taken. Briefly, the surface in each well of
96-well plates was incubated with 200 μL of >99% ethanol for 1 hr
followed by 200 μL of water for over night to move potential
extractables. Finally the surfaces were air dried before sterilization.

[0063]At this point two different acrylate re-plating schedules were
tested: In the first protocol, on day 28, EBs were re-plated on different
acrylic surfaces in 96-well plate or on MATRIGEL-coated wells as positive
control. Seven days later (day 35), cells were fixed with 4% PFA. In the
second protocol, on day 28, EBs were re-plated on MATRIGEL, cultured for
7 days and then (day 35) re-plated on different acrylic surfaces in
96-well plate or on MATRIGEL-coated wells as positive control. Seven days
later (day 42), cells were fixed with 4% PFA.

[0064]Cells from the both protocols were immunostained for OPC-specific
markers, Nestin, Olig1, and counterstained with
4'-6-Diamidino-2-phenylindole DAPI (nuclear stain).

[0065]After scanning each plate with ArrayScan, the following quantitative
analyses were performed for the each surface: 1) TNC: total number of
cells, based on DAPI positive cell number, 2) TNO: total number of OPC,
based on olig1-positive cells.

3. Results

[0066]It was found that only a small portion of the tested surfaces
supported attachment and cell outgrowth from EBs in chemically defined
medium. FIG. 3 are fluorescence images of immunostained hES cell-derived
OPC after re-plating on selected acrylate coated surfaces and positive
control Matrigel surface between 28-day and 35-day for differentiation.
FIG. 4 are fluorescence images of immunostained hES cell-derived OPC
after two re-platings on selected acrylate coated surfaces and positive
control Matrigel surface between 28-day and 35-day, as well as between
35-day and 42-days for differentiation. The immunostaining images showed
that differentiated cells expressed the OPC markers on some acrylate
coating surfaces. The staining of the cells grown on the acrylate
surfaces was similar to the staining observed in the cells grown on the
Martigel control surface. Examples of the coating surfaces which
supported differentiated human OPCs in chemically defined medium are
listed in Table 2, where the volume ratio of monomer (1) to monomer (2)
is 70:30.

[0067]FIGS. 5A-F show images taken from micrographs of hESC derived OPCs
growing on Matrigel® as a positive control and selected embodiments of
surfaces of the present invention; 22-2 (B), 22-3 (C), 133-4 (D), 24-10
(E), and 72-2 (F). FIGS. 5A-F show nuclear staining on hESC derived OPCs
on Matrigel or the above-referenced embodiments of surfaces of the
present invention stained with Hoecst nuclear stain. FIGS. 5A-F
illustrate that embodiments of surfaces of the present invention provide
suitable surfaces to support adhesion and growth of hESC derived OPCs in
chemically defined medium. For other tested homopolymers and copolymer
combinations, that is those combinations that are not listed in Table 2
above, no EB attachment to the surface or cell outgrows from the EBs was
observed.

[0068]Thus, embodiments of SYNTHETIC SURFACES FOR CULTURING STEM CELL
DERIVED OLIGODENDROCYTE PROGENITOR CELLS are disclosed. One skilled in
the art will appreciate that the arrays, compositions, kits and methods
described herein can be practiced with embodiments other than those
disclosed. The disclosed embodiments are presented for purposes of
illustration and not limitation.